Efficient DNA-mediated transformation of Tetrahymena cells has been accomplished by three
methods: microinjection [1], electrotransformation [2] and biolistic bombardment [3]. Both the
germline nucleus (micronucleus) or the somatic nucleus (macronucleus) can be transformed. (Click
here for a description of the two types of nuclei.) Fig. 1 below shows the optimum life cycle stages
for various types of transformation. By the appropriate choice of vectors and conditions, the
incoming plasmid can be autonomously replicated in the MAC for many fissions, or the plasmid
DNA can be inserted at the appropriate chromosome location by exact insert-mediated
homologous recombination. The latter phenomenon allows gene replacements and knockouts,
either in the MAC [ref.4; Fig. 2 below] or the MIC (ref.5; Fig. 3 below). Replacements and
knockout strains are extremely valuable experimental tools, including the testing the indispensability
of cloned genes and isolation of mutants after random mutagenesis of cloned genes. (Click here for
a general Introduction to Tetrahymena Genetics.)

Selection for complete gene replacement by phenotypic assortment enables the distinction between
essential and non-essential genes. The initial transformant contains one transformed gene which has
been disrupted with an expression cassette containing a drug-selectable marker. Its clonal progeny
are serially transferred to higher drug concentrations thus killing cells with fewer copies of the
selectable markers. For a non-essential gene (top diagram), complete replacement is possible. For
an essential gene (bottom diagram), only incompletely assorted cells whose macronuclear copies of
the gene have been partially replaced can be obtained. All arrows indicate multiple generations.

Fig. 3. Gene replacement in the germline; knockout heterokaryons.

Creation and testing of knockout heterokaryons homozygous for disrupted ATU (alpha tubulin)
genes in the micronucleus (deltaATU) and containing only wild type ATU genes in the
macronucleus [4]. Chx is a dominant gene conferring resistance to cycloheximide (cy-r) while the
wild type Chx+ is sensitive (cy-s). Mpr is a dominant gene conferring resistance to 6-methylpurine
(mp-r) to which the wild type Mpr+ gene is sensitive (mp-s). Roman numerals indicate mating
types; mating types not determined are indicated by ?. Crosses (a), (b) and (d) are normal crosses,
while cross (c) is a genomic exclusion cross (see Fig. 4 below for explanation of genomic
exclusion). Note that in cross (d) only a mutant deltaATU/deltaATU Round I product of cross (c)
is shown. An approximately equal number of non-mutant ATU/ATU Round I cells are obtained
that do not yield any pm-r progeny. The genotype of the Mpr locus in Round I exconjugants was
not determined. Values obtained for test crosses (b) and (d) are from a single subline.

Fig. 4. Genomic Exclusion

* ("star") strains, have defective micronuclei; they can form conjugal pairs, but cannot donate
genetic material [Allen,SL (1967) Genetics 55, 797-822]. When mated to a "star" strain, a normal
strain ("left" conjugant, round 1) donates a gametic nucleus, but receives nothing in return. The
single haploid nucleus in each conjugant then diploidizes and most cells separate without forming a
new macronucleus. These cells retain their old (macronuclear) phenotype and are mature (can be
immediately re-mated) but can have new micronuclear genotypes, depending on the genetic
make-up of the normal parent and which meiotic product was (randomly) selected to form gametic
nuclei. This process, referred to as Round 1 of genomic exclusion, greatly facilitates
complementation testing and mutant rescue by cytoplasmic exchange during conjugation. Round 1
genomic exclusion or phenotypic assortment of heterozygotes each allow production of
heterokaryons, cells with different macro- and micronuclear genotypes. Heterokaryons
homozygous or heterozygous for a drug resistance gene in the transcriptionally inert micronucleus
but having only drug-sensitive alleles in the macronucleus are drug sensitive. When these cells
conjugated, the drug sensitive macronucleus is discarded and the drug resistance allele is expressed
in the new macronucleus, allowing simple drug selection for successful mating. Round 1 genomic
exclusion also allows creation of strains homozygous for deleterious or even lethal genes in their
micronuclei and wild type genes in their macronuclei.

Note: The figures and legends in this page were contributed by Dr. Martin Gorovsky, Department
of Biology, University of Rochester, Rochester, NY 14627. Phone: (716)275-6988. Fax:
(716)275-2070. E-mail: micro@dialup.rochester.edu